Therapeutic Effect And Possible Mechanism Of Tongluo Recipe On Diabetic Hepatic Injury

Background and purposeMore and more studies have shown that there is a close link between oxidative stress, diabetes and diabetic complications. The unified theory for pathogenesis of diabetic chronical complications figure out that:Four metabolic pathways have been thought participate in genesis of diabetes mellitus complications, including polyol pathway flux, advanced glycosylation end product formation, protein kinase C pathway and increased hexosamine pathway flux. Now High glucose-induced over-expression of superoxide is considered to be the common mechanism. Diabetic hepatic injury refers to the changes in liver function and histology induced by diabetes, belonging to chronic complications of diabetes mellitus. Oxidative stress and lipid peroxidation, not only can directly damage liver cells and induce inflammatory cells to the chemotactic lipid inflammatory infiltration, but also activate the hepatic fat-storing cells (stellate cells) to promote synthesis of collagen fibers, leading to fibrosis and even cirrhosis of the liver. Antioxidants can reduce the production of free radicals or direct quenching of free radicals produced by the body, enhance the body's antioxidant capacity. Therefore, the use of anti-oxidant treatment of diabetes in order to improve the abnormal pathophysiology, has emerged as one of the important treatments of diabetes and its complications. Many anti-oxidants such as vitamin C, E and a-lipoic acid have beneficial effects on diabetic complications. Tongluo recipe (TLR) is a Chinese herbal compound preparation, from ginseng, leeches, centipedes, Scorpio, eupolyphaga, paeoniae radix, et al. Modern pharmacological studies have shown that the main active ingredients of ginseng ginsenosides with a lipid-lowering, anti-oxidation, anti-apoptotic effect; leeches are diuretic, can eliminate urine protein and improve microcirculation; Eupolyphaga insects and Scorpio have the role of anticoagulant and antithrombosis; paeoniae radix can lower blood concentration, inhibit platelet aggregation and platelet action, decrease fibrinogen, limit thrombosis time.Preliminary study of this topic showed that Tongluo Recipe through its anti-oxidation and improve the blood flow dynamics to reduce kidney, vascular endothelial, myocardial cells, hippocampus damage of diabetic rats. Whether Tongluo Recipe has a therapeutic effect on diabetic hepatic injury, at present there is no relevant reports. In this study, diabetic rats model were induced by streptozotocin(STZ).We observe serum liver function and liver fibrosis-related indicators, oxidative stress and morphological changes in liver tissue of diabetic rats, to explore the therapeutic effect of Tongluo Recipe for diabetic hepatic injury and its possible mechanism.Object of study and methodsExperimental diabetes was induced by intraperitoneal injection of streptozotocin(60 mg/kg)in male Sprague-Dawley rats. The rats with blood glucose levels≥16.7 mmol/L 7 days after injection were included in the study. The study included the following 5 groups:normal control(CN), diabetes mellitus control(DM),low dose TLR (TL)group,middle dose TLR (TM)group and high dose TLR (TH)group,10 rats in each group. Rats received 0.5g·kg-1·d,1.0g·kg-1·d,2.0g·kg-1·d TLR treatment respectively throughout the experimental period of 12 weeks.1. Detection body weight, blood sugar, blood lipids [triglyceride (TG), high-density lipoprotein (HDL)] of all rats.2. Detection serum liver function indexes [alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP)], serum indexes of liver fibrosis [hyaluronic acid (HA), IV collagen (IV-c), laminin (LN),Ⅲprocollagen (PCIII)] of all rats.3. Light microscopy and transmission electron microscope observation of morphological changes in liver tissue of all rats..4. Detection oxidative stress-related indicators in liver tissues[superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), malondialdehyde (MDA)] of all rats.Results:1. Fasting blood glucose and serum triglyceride of diabetic rats were significantly increased (P<0.01), body weights and serum high-density lipoprotein were significantly decreased(P<0.01). After 12 weeks of treatment with Tongluo Recipe, body weights of diabetic rats in mid-dose and high-dose group were increased compared with untreated rats, but there were no statistical significance. Blood glucose and blood lipid indicators have not been improved after Tongluo Recipe treatment.2. Liver damage occurs in diabetic rats. Serum liver function indexes(ALT, AST, and AKP) were significantly increased (P<0.01). After 12 weeks of treatment with Tongluo Recipe, serum ALT, AST levels in mid-dose and high-dose group were significantly decreased (P<0.05).Tongluo Recipe can improve the liver damage to a certain extent.3. The levels of serum liver fibrosis markers (HA andⅣ-c) were significantly increased in diabetic rats (P<0.01). After 12 weeks of treatment with Tongluo Recipe, serum HA and IV-c levels in high-dose group were significantly decreased (P<0.05).4. Light microscopy and transmission electron microscopy observation showed characteristic pathological changes of liver cells in diabetic rats:there were great deal of glycogen granules and increased lipid droplets in cytoplasm, blurred cristae of mitochondria and increased its electron density, rough endoplasmic reticulum localized degranulation, smooth endoplasmic reticulum expanded. Meanwhile, the diabetic rats appeared pathological changes of liver fibrosis and microvascular injury:collagen fibers hyperplasia in hepatic sinusoid and portal area; swelling of mitochondria with cristae loss in vascular endothelial cells, some blood vessel wall thickening, fat-storing cells proliferation in Disse space. After 12 weeks of treatment with Tongluo Recipe, cytoplasmic glycogen granules are obviously decreased, mitochondria cristae become clear and less dense matrix, rough and smooth endoplasmic reticulum near normal.Proliferation of collagen fibers is reduced in hepatic sinusoid and portal area. The morphology of vascular endothelial cells and vascular wall thickness are near normal in portal area. Fat-storing cells are reduced in Disse space. Tongluo Recipe treatment significantly improved the above-mentioned pathological changes.5. Antioxidant enzymes SOD and CAT activities in liver tissue of diabetic rats were significantly decreased (P<0.01); GSH-Px was also decreased, but the difference was not statistically significant; MDA level was significantly increased (P<0.01). Liver tissues were in a state of excessive oxidative stress. Tongluo Recipe has obviously improved SOD activity (P<0.01) in low-dose, mid-dose and high-dose group, and decreased the levels of MDA (P<0.01) in mid-dose and high-dose group. The level of oxidative stress in liver tissue has been partly reduced.Conclusion:1. Serum liver function and liver fibrosis indexes increase in diabetic rats, meanwhile morphological changes of liver tissue are observated. Tongluo recipe can significantly reduce the blood biochemical abnormalities, significantly improve morphological changes of liver. It has a certain therapeutic effect on diabetic hepatic injury with therapeutic dose of2.0g/(kg·d).2. The level of oxidative stress in liver tissue of diabetic rats has been increased, Tongluo recipe can enhance the activity of antioxidant enzyme and remove peroxidation productions. It can decrease the levels of oxidative stress in liver tissues of diabetic rats.3. Glucose and lipid metabolism are significantly disordered in diabetic rats. Tongluo Recipe treatment has no significant effect on lowering blood sugar and blood lipid. Therefore, we believe that the therapeutic effect of Tongluo Recipe on diabetic hepatic injury does not depend on its effect of regulating glucose and lipid levels. Regulation of the level of oxidative stress may be its primary mechanism.